Intravesical BCG has been used successfully to treat superficial bladder cancer for three decades. However, 20% to 30% of patients will fail initial BCG therapy and 30% to 50% of patients will develop recurrent tumors within 5 years. Alternative or complementary strategies for the management of superficial bladder cancer are needed. Interleukin-12 (IL-12) is a potent TH1 cytokine with robust antitumor activity and the ability to potentiate immunologic memory. Unfortunately, intravesical IL-12 did not show antitumor efficacy in a recent clinical study of patients with recurrent superficial bladder cancer. We hypothesized that coformulation of IL-12 with chitosan, a biocompatible, mucoadhesive polysaccharide, could improve intravesical IL-12 delivery and provide an effective and durable alternative for the treatment of superficial bladder cancer. In antitumor studies, 88% to 100% of mice bearing orthotopic bladder tumors were cured after four intravesical treatments with chitosan/IL-12. In contrast, only 38% to 60% of mice treated with IL-12 alone and 0% treated with BCG were cured. Antitumor responses following chitosan/ IL-12 treatments were durable and provided complete protection from intravesical tumor rechallenge. Urinary cytokine analysis showed that chitosan/IL-12 induced multiple TH1 cytokines at levels significantly higher than either IL-12 alone or BCG. Immunohistochemistry revealed moderate to intense tumor infiltration by T cells and macrophages following chitosan/IL-12 treatments. Bladder submucosa from cured mice contained residual populations of immune cells that returned to baseline levels after several months. Intravesical chitosan/IL-12 is a well-tolerated, effective immunotherapy that deserves further consideration for testing in humans for the management of superficial bladder cancer. IL-12 is a potent antitumor cytokine that exhibits significant clinical toxicities after systemic administration. We hypothesized that intratumoral (i.t.) administration of IL-12 coformulated with the biodegradable polysaccharide chitosan could enhance the antitumor activity of IL-12 while limiting its systemic toxicity. Noninvasive imaging studies monitored local retention of IL-12, with and without chitosan coformulation, after i.t. injection. Antitumor efficacy of IL-12 alone and IL-12 coformulated with chitosan (chitosan/IL-12) was assessed in mice bearing established colorectal (MC32a) and pancreatic (Panc02) tumors. Additional studies involving depletion of immune cell subsets, tumor rechallenge, and CTL activity were designed to elucidate mechanisms of regression and tumor-specific immunity. Coformulation with chitosan increased local IL-12 retention from 1 to 2 days to 5 to 6 days. Weekly i.t. injections of IL-12 alone eradicated less than 10% of established MC32a and Panc02 tumors, while i.t. chitosan/IL-12 immunotherapy caused complete tumor regression in 80% to 100% of mice. Depletion of CD4+ or Gr-1+ cells had no impact on chitosan/IL-12-mediated tumor regression. However, CD8+ or NK cell depletion completely abrogated antitumor activity. I.t. chitosan/IL-12 immunotherapy generated systemic tumor-specific immunity, as greater than 80% of mice cured with i.t. chitosan/IL-12 immunotherapy were at least partially protected from tumor rechallenge. Furthermore, CTLs from spleens of cured mice lysed MC32a and gp70 peptide-loaded targets. Chitosan/IL-12 immunotherapy increased local retention of IL-12 in the tumor microenvironment, eradicated established, aggressive murine tumors, and generated systemic tumor-specific protective immunity. Chitosan/IL-12 is a well-tolerated, effective immunotherapy with considerable potential for clinical translation.